Propylene glycol-containing peptide formulations which are optimal for production and for use in injection devices

ABSTRACT

The present invention relates to pharmaceutical formulations comprising a peptide and propylene glycol, to methods of preparing such formulations, and to uses of such formulations in the treatment of diseases and conditions for which use of the peptide contained in such formulations is indicated. The present invention further relates to methods for reducing the clogging of injection devices by a peptide formulation and for reducing deposits on production equipment during production of a peptide formulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/435,977, filed May 17, 2006, which is a continuation of InternationalApplication serial no. PCT/DK2004/000792 filed Nov. 18, 2004 and claimspriority from U.S. Application Ser. No. 60/524,653 filed Nov. 24, 2003and from Danish Application serial no. PA 2003 01719 filed Nov. 20,2003.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical formulations comprisinga peptide and propylene glycol, to methods of preparing suchformulations, and to uses of such formulations in the treatment ofdiseases and conditions for which use of the peptide contained in suchformulations is indicated. The present invention further relates tomethods for reducing the clogging of injection devices by a peptideformulation and for reducing deposits on production equipment duringproduction of a peptide formulation.

BACKGROUND OF THE INVENTION

The inclusion of isotonicity agents in peptide-containing pharmaceuticalformulations is widely known and one of the more common isotonic agentsused in such formulations is mannitol. However, the present inventorshave observed that mannitol causes problems during the production ofpeptide formulations as it crystallizes resulting in deposits in theproduction equipment and in the final product. Such deposits increasethe need to clean the filling equipment during production of theformulation and this results in reduced production capability. Inaddition, such deposits may also result in reduced yield of the finalproduct since vials/cartridges containing the peptide formulation mayneed to be discarded if particles are present. Finally, the presentinventors have observed that in peptide formulations to be administeredby injection, the presence of mannitol results in clogging of injectiondevices.

Accordingly, it is desirable to identify an alternative isotonic agentto mannitol for inclusion in peptide-containing formulations and inparticular, for inclusion in peptide formulations which are administeredby injection.

SUMMARY OF THE INVENTION

The present inventors have discovered that peptide formulationscontaining propylene glycol at certain concentrations exhibit reduceddeposits in production equipment and in the final product and alsoexhibit reduced clogging of injection devices. The present compositionsmay be formulated with any peptide and are also physically andchemically stable thus rendering them shelf-stable and suitable forinvasive (eg. injection, subcutaneous injection, intramuscular,intravenous or infusion) as well as non-invasive (eg nasal, oral,pulmonary, transdermal or transmucosal e.g. buccal) means ofadministration.

The present invention therefore relates to a pharmaceutical formulationcomprising a peptide and propylene glycol, where the propylene glycol ispresent in a concentration of 1-100 mg/ml and the pH of the formulationis from 7-10. In a preferred embodiment, the pharmaceutical formulationsof the invention further contain a buffer and a preservative.

The present invention also relates to methods for producing thepharmaceutical formulations of the invention.

In one embodiment, the method for preparing a peptide formulationcomprises:

-   -   a) preparing a first solution by dissolving preservative,        propylene glycol and buffer in water;    -   b) preparing a second solution by dissolving the peptide in        water;    -   c) mixing the first and second solutions; and    -   d) adjusting the pH of the mixture in c) to the desired pH.

In another embodiment, the method for preparing a peptide formulationcomprises:

-   -   a) preparing a first solution by dissolving preservative and        buffer in water;    -   b) adding propylene glycol to the first solution;    -   c) mixing the first solution with a second solution containing        peptide dissolved in water; and    -   d) adjusting the pH of the mixture in c) to the desired pH.

In yet another embodiment, the method for preparing a peptideformulation comprises:

-   -   a) preparing a solution by dissolving preservative, buffer and        propylene glycol in water;    -   b) adding the peptide to the solution of step a); and    -   c) adjusting the pH of the solution of step b) to the desired        pH.

The present invention further relates to methods of treatment using thepharmaceutical formulations of the invention where the compositions areadministered in an amount effective to combat the disease, condition, ordisorder for which administration of the peptide contained in theformulation is indicated.

In addition the present invention also relates to a method for reducingdeposits on production equipment during production of a peptideformulation, where the method comprises replacing the isotonicity agentpreviously utilized in said formulation with propylene glycol at aconcentration of between 1-100 mg/ml.

In one embodiment, the reduction in deposits on the production equipmentduring production by the propylene glycol-containing formulationrelative to that observed for the formulation containing the previouslyutilized isotonicity agent is measured by a simulated fillingexperiment.

The present invention also relates to a method for reducing deposits inthe final product during production of a peptide formulation, where themethod comprises replacing the isotonicity agent previously utilized insaid formulation with propylene glycol at a concentration of between1-100 mg/ml.

In one embodiment, the reduction in deposits in the final product ismeasured by a reduction in the number of vials and/or cartridges of thepropylene glycol-containing formulation that must be discarded due todeposits relative to number of vials and/or cartridges of theformulation containing the previously utilized isotonicity agent thatmust be discarded due to deposits.

The present invention further relates to a method for reducing theclogging of injection devices by a peptide formulation, where the methodcomprises replacing the isotonicity agent previously utilized in saidformulation with propylene glycol at a concentration of between 1-100mg/ml.

In one embodiment, the reduction in clogging of the injection device bythe propylene glycol-containing formulation relative to that observedfor the formulation containing the previously utilized isotonicity agentis measured in a simulated in use study.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a photograph of dried droplets on microscope slides of fromleft to right, placebo (no peptide) formulations containing no isotonicagent (e only water, preservative and buffer), mannitol, sorbitol,xylitol, sucrose or glycerol as the isotonic agent with the far rightslide containing mannitol with peptideArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37).

FIG. 2 shows light microscopy pictures of from left to right, some ofthe dried droplets of placebo formulations containing mannitol, arginin,inositol or glycerol as the isotonic agent.

FIG. 3 shows light microscopy pictures of clogged needles dosed withplacebo formulations containing myoinositol, maltose or glycerol as theisotonic agent.

FIG. 4 shows light microscopy pictures of deposits on needles dosed withplacebo formulations containing glycine, lactose or mannitol as theisotonic agent.

FIG. 5 shows filling equipment after 24 hours simulated filling withArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) mediumcontaining myo-inositol.

FIG. 6 shows deposits on filling equipment after 24 hours simulatedfilling with a mannitol-containing placebo formulation.

FIG. 7 shows deposits on needles dosed with mannitol (top panel) andpropylene glycol (bottom panel)-containingArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) formulations.

DESCRIPTION OF THE INVENTION

The present invention relates to a pharmaceutical formulation comprisinga peptide or a mixture of peptides and propylene glycol where the finalconcentration of propylene glycol in the formulation is 1-100 mg/ml andthe pH of the formulation is in the range of from 7-10.

The pharmaceutical formulations of the invention are found to be optimalfor production because they exhibit reduced deposits in productionequipment relative to formulations containing other isotonicity agentsas measured by the simulated filling studies described in the Examples.In addition, the pharmaceutical formulations of the invention are foundto be optimal for use in injection devices because they exhibit reducedclogging of the injection devices relative to formulations containingother isotonicity agents as measured by the simulated in use studiesdescribed in the Examples.

The formulations of the present invention may be formulated with anypeptide where examples of such peptides include, but are not limited to,glucagon, human growth hormone (hGH), insulin, aprotinin, FactorVII,tissue plasminogen activator (TPA), FactorVIIa, FFR-FactorVIIa,heparinase, ACTH, Heparin Binding Protein, corticotropin-releasingfactor, angiotensin, calcitonin, glucagon-like peptide-1, glucagon-likepeptide-2, insulin-like growth factor-1, insulin-like growth factor-2,fibroblast growth factors, gastric inhibitory peptide, growthhormone-releasing factor, pituitary adenylate cyclase activatingpeptide, secretin, enterogastrin, somatostatin, somatomedin, parathyroidhormone, thrombopoietin, erythropoietin, hypothalamic releasing factors,prolactin, thyroid stimulating hormones, endorphins, enkephalins,vasopressin, oxytocin, opiods, DPP IV, interleukins, immunoglobulins,complement inhibitors, serine protease inhibitors, cytokines, cytokinereceptors, PDGF, tumor necrosis factors, tumor necrosis factorsreceptors, growth factors and analogues as well as derivatives thereofwhere each of these peptides constitutes an alternative embodiment ofthe present invention.

In the present application, the designation “an analogue” is used todesignate a peptide wherein one or more amino acid residues of theparent peptide have been substituted by another amino acid residueand/or wherein one or more amino acid residues of the parent peptidehave been deleted and/or wherein one or more amino acid residues havebeen added to the parent peptide. Such addition can take place either atthe N-terminal end or at the C-terminal end of the parent peptide orboth. Typically “an analogue” is a peptide wherein 6 or less amino acidshave been substituted and/or added and/or deleted from the parentpeptide, more preferably a peptide wherein 3 or less amino acids havebeen substituted and/or added and/or deleted from the parent peptide,and most preferably, a peptide wherein one amino acid has beensubstituted and/or added and/or deleted from the parent peptide.

In the present application, “a derivative” is used to designate apeptide or analogue thereof which is chemically modified by introducingan organic substituent e.g. ester, alkyl or lipophilic functionalities,on one or more amino acid residues of the peptide or analogue thereof.

In one embodiment, the peptide to be included in the formulation of theinvention is a GLP-1 agonist where “a GLP-1 agonist” is understood torefer to any peptide which fully or partially activates the human GLP-1receptor. In a preferred embodiment, the “GLP-1 agonist” is any peptidethat binds to a GLP-1 receptor, preferably with an affinity constant(K_(D)) or a potency (EC₅₀) of below 1 μM, e.g. below 100 nM as measuredby methods known in the art (see e.g. WO 98/08871) and exhibitsinsulinotropic activity, where insulinotropic activity may be measuredin vivo or in vitro assays known to those of ordinary skill in the art.For example, the GLP-1 agonist may be administered to an animal and theinsulin concentration measured over time.

Methods for identifying GLP-1 agonists are described in WO 93/19175(Novo Nordisk NS) and examples of suitable GLP-1 analogues andderivatives which can be used according the present invention includesthose referred to in WO 99/43705 (Novo Nordisk A/S), WO 99/43706 (NovoNordisk A/S), WO 99/43707 (Novo Nordisk A/S), WO 98/08871 (analogueswith lipophilic substituent) and in WO 02/46227 (analogues fused toserum albumin or to Fc portion of an Ig). (Novo Nordisk A/S), WO99/43708 (Novo Nordisk A/S), WO 99/43341 (Novo Nordisk A/S), WO 87/06941(The General Hospital Corporation), WO 90/11296 (The General HospitalCorporation), WO 91/11457 (Buckley et al.), WO 98/43658 (Eli Lilly &Co.), EP 0708179-A2 (Eli Lilly & Co.), EP 0699686-A2 (Eli Lilly & Co.),WO 01/98331 (Eli Lilly & Co).

In one embodiment, the GLP-1 agonist is selected from the groupconsisting of GLP-1(7-36)-amide, GLP-1(7-37), a GLP-1(7-36)-amideanalogue, a GLP-1(7-37) analogue, or a derivative of any of these.

In one embodiment, the GLP-1 agonist is a derivative ofGLP-1(7-36)-amide, GLP-1(7-37), a GLP-1(7-36)-amide analogue or aGLP-1(7-37) analogue, which comprises a lipophilic substituent.

In this embodiment of the invention, the GLP-1 derivative preferably hasthree lipophilic substituents, more preferably two lipophilicsubstituents, and most preferably one lipophilic substituent attached tothe parent peptide (ie GLP-1(7-36)-amide, GLP-1(7-37), aGLP-1(7-36)-amide analogue or a GLP-1(7-37) analogue), where eachlipophilic substituent(s) preferably has 4-40 carbon atoms, morepreferably 8-30 carbon atoms, even more preferably 8-25 carbon atoms,even more preferably 12-25 carbon atoms, and most preferably 14-18carbon atoms.

In one embodiment, the lipophilic substituent comprises a partially orcompletely hydrogenated cyclopentanophenathrene skeleton.

In another embodiment, the lipophilic substituent is a straight-chain orbranched alkyl group.

In yet another embodiment, the lipophilic substituent is an acyl groupof a straight-chain or branched fatty acid. Preferably, the lipophilicsubstituent is an acyl group having the formula CH₃(CH₂)₂₀CO—, wherein nis an integer from 4 to 38, preferably an integer from 12 to 38, andmost preferably is CH₃(CH₂)₁₂CO—, CH₃(CH₂)₁₄CO—, CH₃(CH₂)₁₆CO—,CH₃(CH₂)₁₈CO—, CH₃(CH₂)₂₀CO— and CH₃(CH₂)₂₂CO—. In a more preferredembodiment, the lipophilic substituent is tetradecanoyl. In a mostpreferred embodiment, the lipophilic substituent is hexadecanoyl.

In a further embodiment of the present invention, the lipophilicsubstituent has a group which is negatively charged such as a carboxylicacid group. For example, the lipophilic substituent may be an acyl groupof a straight-chain or branched alkane α,ω-dicarboxylic acid of theformula HOOC(CH₂)_(m)CO—, wherein m is an integer from 4 to 38,preferably an integer from 12 to 38, and most preferably isHOOC(CH₂)₁₄CO—, HOOC(CH₂)₁₆CO—, HOOC(CH₂)₁₈CO—, HOOC(CH₂)₂₀CO— orHOOC(CH₂)₂₂CO—.

In the GLP-1 derivatives of the invention, the lipophilic substituent(s)contain a functional group which can be attached to one of the followingfunctional groups of an amino acid of the parent GLP-1 peptide:

(a) the amino group attached to the alpha-carbon of the N-terminal aminoacid,

(b) the carboxy group attached to the alpha-carbon of the C-terminalamino acid,

(c) the epsilon-amino group of any Lys residue,

(d) the carboxy group of the R group of any Asp and Glu residue,

(e) the hydroxy group of the R group of any Tyr, Ser and Thr residue,

(f) the amino group of the R group of any Trp, Asn, Gln, Arg, and Hisresidue, or

(g) the thiol group of the R group of any Cys residue.

In one embodiment, a lipophilic substituent is attached to the carboxygroup of the R group of any Asp and Glu residue.

In another embodiment, a lipophilic substituent is attached to thecarboxy group attached to the alpha-carbon of the C-terminal amino acid.

In a most preferred embodiment, a lipophilic substituent is attached tothe epsilon-amino group of any Lys residue.

In a preferred embodiment of the invention, the lipophilic substituentis attached to the parent GLP-1 peptide by means of a spacer. A spacermust contain at least two functional groups, one to attach to afunctional group of the lipophilic substituent and the other to afunctional group of the parent GLP-1 peptide.

In one embodiment, the spacer is an amino acid residue except Cys orMet, or a dipeptide such as Gly-Lys. For purposes of the presentinvention, the phrase “a dipeptide such as Gly-Lys” means anycombination of two amino acids except Cys or Met, preferably a dipeptidewherein the C-terminal amino acid residue is Lys, His or Trp, preferablyLys, and the N-terminal amino acid residue is Ala, Arg, Asp, Asn, Gly,Glu, Gln, Ile, Leu, Val, Phe, Pro, Ser, Tyr, Thr, Lys, His and Trp.Preferably, an amino group of the parent peptide forms an amide bondwith a carboxylic group of the amino acid residue or dipeptide spacer,and an amino group of the amino acid residue or dipeptide spacer formsan amide bond with a carboxyl group of the lipophilic substituent.

Preferred spacers are lysyl, glutamyl, asparagyl, glycyl, beta-alanyland gamma-aminobutanoyl, each of which constitutes an individualembodiment. Most preferred spacers are glutamyl and beta-alanyl. Whenthe spacer is Lys, Glu or Asp, the carboxyl group thereof may form anamide bond with an amino group of the amino acid residue, and the aminogroup thereof may form an amide bond with a carboxyl group of thelipophilic substituent. When Lys is used as the spacer, a further spacermay in some instances be inserted between the ε-amino group of Lys andthe lipophilic substituent. In one embodiment, such a further spacer issuccinic acid which forms an amide bond with the ε-amino group of Lysand with an amino group present in the lipophilic substituent. Inanother embodiment such a further spacer is Glu or Asp which forms anamide bond with the ε-amino group of Lys and another amide bond with acarboxyl group present in the lipophilic substituent, that is, thelipophilic substituent is a N⁸-acylated lysine residue.

In another embodiment, the spacer is an unbranched alkaneα,ω-dicarboxylic acid group having from 1 to 7 methylene groups, whichspacer forms a bridge between an amino group of the parent peptide andan amino group of the lipophilic substituent. Preferably, the spacer issuccinic acid.

In a further embodiment, the lipophilic substituent with the attachedspacer is a group of the formula CH₃(CH₂)_(p)NH—CO(CH₂)_(q)CO—, whereinp is an integer from 8 to 33, preferably from 12 to 28 and q is aninteger from 1 to 6, preferably 2.

In a further embodiment, the lipophilic substituent with the attachedspacer is a group of the formula CH₃(CH₂)_(r)CO—NHCH(COOH)(CH₂)₂CO—,wherein r is an integer from 4 to 24, preferably from 10 to 24.

In a further embodiment, the lipophilic substituent with the attachedspacer is a group of the formula CH₃(CH₂)_(s)CO—NHCH((CH₂)₂COOH)CO—,wherein s is an integer from 4 to 24, preferably from 10 to 24.

In a further embodiment, the lipophilic substituent is a group of theformula COOH(CH₂)_(t)CO— wherein t is an integer from 6 to 24.

In a further embodiment, the lipophilic substituent with the attachedspacer is a group of the formula —NHCH(COOH)(CH₂)₄NH—CO(CH₂)_(u)CH₃,wherein u is an integer from 8 to 18.

In a further embodiment, the lipophilic substituent with the attachedspacer is a group of the formula CH₃(CH₂)_(v)CO—NH—(CH₂)_(z)—CO, whereinv is an integer from 4 to 24 and z is an integer from 1 to 6.

In a further embodiment, the lipophilic substituent with the attachedspacer is a group of the formula—NHCH(COOH)(CH₂)₄NH—COCH((CH₂)₂COOH)NH—CO(CH₂)_(w)CH₃, wherein w is aninteger from 10 to 16.

In a further embodiment, the lipophilic substituent with the attachedspacer is a group of the formula—NHCH(COOH)(CH₂)₄NH—CO(CH₂)₂CH(COOH)NHCO(CH₂)_(x)CH₃, wherein x is zeroor an integer from 1 to 22, preferably 10 to 16.

In yet another embodiment the GLP-1 agonist isArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37).

In yet another embodiment the GLP-1 agonist is selected from the groupconsisting of Gly⁸-GLP-1(7-36)-amide, Gly⁸-GLP-1(7-37),Val⁸-GLP-1(7-36)-amide, Val⁸-GLP-1(7-37), Val⁸Asp²²-GLP-1(7-36)-amide,Val⁸Asp²²-GLP-1(7-37), Val⁸Glu²²-GLP-1(7-36)-amide,Val⁸Glu²²-GLP-1(7-37), Val⁸Lys²²-GLP-1(7-36)-amide,Val⁸Lys²²-GLP-1(7-37), Val⁸Arg²²-GLP-1(7-36)-amide,Val⁸Arg²²-GLP-1(7-37), Val⁸His²²-GLP-1(7-36)-amide,Val⁸His²²-GLP-1(7-37), analogues thereof and derivatives of any ofthese.

In yet another embodiment the GLP-1 agonist is selected from the groupconsisting of Arg²⁶-GLP-1(7-37); Arg³⁴-GLP-1(7-37); Lys³⁶-GLP-1(7-37);Arg^(26,34)Lys³⁶-GLP-1(7-37); Arg^(26,34)-GLP-1(7-37);Arg^(26,34)Lys⁴⁶-GLP-1(7-37); Arg²⁶Lys³⁶-GLP-1(7-37);Arg³⁴Lys³⁶-GLP-1(7-37); Val⁸Arg²²-GLP-1(7-37); Met⁸Arg²²-GLP-1(7-37);Gly⁸His²²-GLP-1(7-37); Val⁸His²²-GLP-1(7-37); Met⁸His²²-GLP-1(7-37);His³⁷-GLP-1(7-37); Gly⁸-GLP-1(7-37); Val⁸-GLP-1(7-37); Met⁸-GLP-1(7-37);Gly⁸Asp²²-GLP-1(7-37); Val⁸Asp²²-GLP-1(7-37); Met⁸Asp²²-GLP-1(7-37);Gly⁸Glu²²-GLP-1(7-37); Val⁸Glu²²-GLP-1(7-37); Met⁸Glu²²-GLP-1(7-37);Gly⁸Lys²²-GLP-1(7-37); Val⁸Lys²²-GLP-1(7-37); Met⁸Lys²²-GLP-1(7-37);Gly⁸Arg²²-GLP-1(7-37); Val⁸Lys²²His³⁷-GLP-1(7-37);Gly⁸Glu²²His³⁷-GLP-1(7-37); Val⁸Glu²²His³⁷-GLP-1(7-37);Met⁸Glu²²His³⁷-GLP-1(7-37); Gly⁸Lys²² His³⁷-GLP-1(7-37);Met⁸Lys²²His³⁷-GLP-1(7-37); Gly⁸Arg²²His³⁷-GLP-1(7-37);Val⁸Arg²²His³⁷-GLP-1(7-37); Met⁸Arg²²His³⁷-GLP-1(7-37);Gly⁸His²²His³⁷-GLP-1(7-37); Val⁸His²²His³⁷-GLP-1(7-37);Met⁸His²²His³⁷-GLP-1(7-37); Gly⁸His³⁷-GLP-1(7-37);Val⁸His³⁷-GLP-1(7-37); Met⁸His³⁷-GLP-1(7-37); Gly⁸Asp²²His³⁷-GLP-1(7-37); Val⁸Asp²²His³⁷-GLP-1(7-37);Met⁸Asp²²His³⁷-GLP-1(7-37); Arg²⁶-GLP-1(7-36)-amide;Arg³⁴-GLP-1(7-36)-amide; Lys³⁶-GLP-1(7-36)-amide;Arg^(26,34)Lys³⁶-GLP-1(7-36)-amide; Arg^(26,34)-GLP-1(7-36)-amide;Arg^(26,34)Lys⁴¹³-GLP-1(7-36)-amide; Arg²⁶Lys³⁶-GLP-1(7-36)-amide;Arg³⁴Lys³⁶-GLP-1(7-36)-amide; Gly⁸-GLP-1(7-36)-amide;Val⁸-GLP-1(7-36)-amide; Met⁸-GLP-1(7-36)-amide;Gly⁸Asp²²-GLP-1(7-36)-amide; Gly⁸Glu²²His³⁷-GLP-1(7-36)-amide;Val⁸Asp²²-GLP-1(7-36)-amide; Met⁸Asp²²-GLP-1(7-36)-amide;Gly⁸Glu²²-GLP-1(7-36)-amide; Val⁸Glu²²-GLP-1(7-36)-amide;Met⁸Glu²²-GLP-1(7-36)-amide; Gly⁸Lys²²-GLP-1(7-36)-amide;Val⁸Lys²²-GLP-1(7-36)-amide; Met⁸Lys²²-GLP-1(7-36)-amide;Gly⁸His²²His³⁷-GLP-1(7-36)-amide; Gly⁸Arg²²-GLP-1(7-36)-amide;Val⁸Arg²²-GLP-1(7-36)-amide; Met⁸Arg²²-GLP-1(7-36)-amide;Gly⁸His²²-GLP-1(7-36)-amide; Val⁸His²²-GLP-1(7-36)-amide;Met⁸His²²-GLP-1(7-36)-amide; His³⁷-GLP-1(7-36)-amide;Val⁸Arg²²His³⁷-GLP-1(7-36)-amide; Met⁸Arg²²His³⁷-GLP-1(7-36)-amide;Gly⁸His³⁷-GLP-1(7-36)-amide; Val⁸His³⁷-GLP-1(7-36)-amide;Met⁸His³⁷-GLP-1(7-36)-amide; Gly⁸Asp²² His³⁷-GLP-1(7-36)-amide;Val⁸Asp²²His³⁷-GLP-1(7-36)-amide; Met⁸Asp²²His³⁷-GLP-1(7-36)-amide;Val⁸Glu²²His³⁷-GLP-1(7-36)-amide; Met⁸Glu²²His³⁷-GLP-1(7-36)-amide;Gly⁸Lys²² His³⁷-GLP-1(7-36)-amide; Val⁸Lys²²His³⁷-GLP-1(7-36)-amide;Met⁸Lys²²His³⁷-GLP-1(7-36)-amide; Gly⁸Arg²²His³⁷-GLP-1(7-36)-amide;Val⁸His²²His³⁷-GLP-1(7-36)-amide; Met⁸His²²His³⁷-GLP-1(7-36)-amide; andderivatives thereof.

In yet another embodiment the GLP-1 agonist is selected from the groupconsisting of Val⁸Trp¹⁹Glu²²-GLP-1(7-37), Val⁸Glu²²Val²⁵-GLP-1(7-37),Val⁸Tyr¹⁶Glu²²-GLP-1(7-37), Val⁸Trp¹⁶Glu²²-GLP-1(7-37),Val⁸Leu¹⁶Glu²²-GLP-1(7-37), Val⁸Tyr¹⁸Glu²²-GLP-1(7-37),Val⁸Glu²²His³⁷-GLP-1(7-37), Val⁸Glu²²Ile³³-GLP-1(7-37),Val⁸Trp¹⁶Glu²²Val²⁵Ile³³-GLP-1(7-37), Val⁸Trp¹⁶Glu²²Ile³³-GLP-1(7-37),Val⁸Glu²²Val²⁵Ile³³-GLP-1(7-37), Val⁸Trp¹⁶Glu²²Val²⁵-GLP-1(7-37),analogues thereof and derivatives of any of these.

In yet another embodiment the GLP-1 agonist is exendin-4 or exendin-3,an exendin-4 or exendin-3 analogue or a derivative of any of these.

Examples of exendins as well as analogues, derivatives, and fragmentsthereof to be included within the present invention are those disclosedin WO 97/46584, U.S. Pat. No. 5,424,286 and WO 01/04156. U.S. Pat. No.5,424,286 describes a method for stimulating insulin release with anexendin polypeptide. The exendin polypeptides disclosed includeHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX; wherein X=P or Y, andHX1X2GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS; wherein X1×2=SD (exendin-3)or GE (exendin-4)). WO 97/46584 describes truncated versions of exendinpeptide(s). The disclosed peptides increase secretion and biosynthesisof insulin, but reduce those of glucagon. WO 01/04156 describesexendin-4 analogues and derivatives as well as the preparation of thesemolecules. Exendin-4 analogues stabilized by fusion to serum albumin orFc portion of an Ig are disclosed in WO 02/46227.

In one embodiment, the exendin-4 analogue isHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-amide.

Where the peptide to be included in the formulation of the invention isa GLP-1 agonist, the GLP-1 agonist is present in a concentration fromabout 0.1 mg/ml to about 100 mg/ml, more preferably in a concentrationfrom about 0.1 mg/ml to about 50 mg/ml, and most preferably in aconcentration of from about 0.1 mg/ml to about 10 mg/ml.

In another embodiment, the peptide to be included in the formulation ofthe invention is insulin, where “insulin” is understood to mean humaninsulin, [where “human insulin” means insulin having the amino acidsequence shown in D S H W Nicol and L F Smith: Nature, (1960)4736:483-485, which is hereby incorporated by reference], human insulinanalogs, human insulin derivatives or mixtures thereof, where examplesof insulin analogs and derivatives are those disclosed in EP 0 792 290(Novo Nordisk A/S), EP 0 214 826 and EP 0 705 275 (Novo Nordisk A/S),U.S. Pat. No. 5,504,188 (Eli Lilly), EP 0 368 187 (Aventis), U.S. Pat.Nos. 5,750,497 and 6,011,007, EP 375437 and EP 383472 and where suchinsulins may include, but are not limited to, NPH insulin, Lys β29(Nε-tetradecanoyl) des(B30) human insulin,Lys^(B29)-(N^(ε)-(γ-glutamyl-N^(α)-lithocholyl) des(B30) human insulin,N^(□B29)-octanoyl insulin, 30/70 mixtures of prompt insulin zinc(SemiLente®) with extended insulin zinc (Ultralente®), sold commerciallyas Lente®, insulin glargine (Lantus®) or extended insulin zinc(Ultralente®), Lys^(B28) Pro^(B29) human insulin (Humalog®), Asp^(B28)human insulin, insulin aspart (Novolog®), or a 30/70 mixture of insulinaspart and insulin aspart protamine (NovoMix®).

In one embodiment, the insulin is a derivative of human insulin or ahuman insulin analogue where the derivative contains at least one lysineresidue and a lipophilic substituent is attached to the epsilon aminogroup of the lysine residue.

In one embodiment, the lysine residue to which the lipophilicsubstituent is attached is present at position B28 of the insulinpeptide.

In an alternative embodiment, the lysine residue to which the lipophilicsubstituent is attached is present at position B29 of the insulinpeptide.

In yet another embodiment, lipophilic substituent is an acyl groupcorresponding to a carboxylic acid having at least 6 carbon atoms.

In another preferred embodiment, the lipophilic substituent is an acylgroup, branched or unbranched, which corresponds to a carboxylic acidhaving a chain of carbon atoms 8 to 24 atoms long.

In another preferred embodiment, the lipophilic substituent is an acylgroup corresponding to a fatty acid having at least 6 carbon atoms.

In another preferred embodiment, the lipophilic substituent is an acylgroup corresponding to a linear, saturated carboxylic acid having from 6to 24 carbon atoms.

In another preferred embodiment, the lipophilic substituent is an acylgroup corresponding to a linear, saturated carboxylic acid having from 8to 12 carbon atoms.

In another preferred embodiment, the lipophilic substituent is an acylgroup corresponding to a linear, saturated carboxylic acid having from10 to 16 carbon atoms.

In another preferred embodiment, the lipophilic substituent is an oligooxyethylene group comprising up to 10, preferably up to 5, oxyethyleneunits.

In another preferred embodiment, the lipophilic substituent is an oligooxypropylene group comprising up to 10, preferably up to 5, oxypropyleneunits.

In one preferred embodiment, the invention relates to a human insulinderivative in which the B30 amino acid residue is deleted or is anyamino acid residue which can be coded for by the genetic code exceptLys, Arg and Cys; the A21 and the B3 amino acid residues are,independently, any amino acid residues which can be coded for by thegenetic code except Lys, Arg and Cys; Phe^(B1) may be deleted; the□-amino group of Lys^(B29) has a lipophilic substituent which comprisesat least 6 carbon atoms; and 2-4 Zn²⁺ ions may be bound to each insulinhexamer with the proviso that when B30 is Thr or Ala and A21 and B3 areboth Asn, and Phe^(B1) is not deleted, then 2-4 Zn²⁺ ions are bound toeach hexamer of the insulin derivative.

In another preferred embodiment, the invention relates to a humaninsulin derivative in which the B30 amino acid residue is deleted or isany amino acid residue which can be coded for by the genetic code exceptLys, Arg and Cys; the A21 and the B3 amino acid residues are,independently, any amino acid residues which can be coded for by thegenetic code except Lys, Arg and Cys, with the proviso that if the B30amino acid residue is Ala or Thr, then at least one of the residues A21and B3 is different from Asn; Phe^(B1) may be deleted; and the □-aminogroup of Lys^(B29) has a lipophilic substituent which comprises at least6 carbon atoms.

In another preferred embodiment, the invention relates to a humaninsulin derivative in which the B30 amino acid residue is deleted or isany amino acid residue which can be coded for by the genetic code exceptLys, Arg and Cys; the A21 and the B3 amino acid residues are,independently, any amino acid residues which can be coded for by thegenetic code except Lys, Arg and Cys; Phe^(B1) may be deleted; the□-amino group of Lys^(B29) has a lipophilic substituent which comprisesat least 6 carbon atoms; and 2-4 Zn²⁺ ions are bound to each insulinhexamer.

Where the peptide to be included in the formulation of the invention isan insulin, the insulin is present in a concentration from about 0.5mg/ml to about 20 mg/ml, more preferably in a concentration from about 1mg/ml to about 15 mg/ml.

In another embodiment, the peptide to be included in the formulations ofthe invention is hGH or Met-hGH.

Where the peptide to be included in the formulation of the invention ishGH or Met-hGH, the hGH or Met-hGH is present in a concentration fromabout 0.5 mg/ml to about 50 mg/ml, more preferably in a concentrationfrom about 1 mg/ml to about 10 mg/ml.

In yet another embodiment, the peptide to be included in theformulations of the invention is GLP-2 or an analogue or derivativethereof.

Where the peptide to be included in the formulation of the invention isGLP-2 or an analogue or derivative thereof, the GLP-2 or an analogue orderivative thereof is present in a concentration from about 1 mg/ml toabout 100 mg/ml, more preferably in a concentration from about 1 mg/mlto about 10 mg/ml.

In yet a further embodiment, the peptide to be included in theformulations of the invention is Factor VII or Factor VIIa or ananalogue or derivative thereof.

Where the peptide to be included in the formulation of the invention isFactor VII or Factor VIIa or an analogue or derivative thereof, theFactor VII or Factor VIIa or an analogue or derivative thereof ispresent in a concentration from about 0.1 mg/ml to about 10 mg/ml, morepreferably in a concentration from about 0.5 mg/ml to about 5 mg/ml.

In one embodiment, the final concentration of propylene glycol in theformulations of the invention is from about 1 to about 50 mg/ml.

In another embodiment, the final concentration of propylene glycol inthe formulations of the invention is from about 5 to about 25 mg/ml.

In yet another embodiment, the final concentration of propylene glycolin the formulations of the invention is from about 8 to about 16 mg/ml.

In yet a further embodiment, the final concentration of propylene glycolin the formulations of the invention is from about 13 to about 15 mg/ml.

In still another embodiment, the final concentration of propylene glycolin the formulations of the invention is from about 13.5 to about 14.5mg/ml.

In another embodiment of the invention, the formulation has a pH in therange from about 7.0 to about 9.5 where the term “about” as used inconnection with pH means + or −0.1 pH units from the stated number.

In a further embodiment of the invention, the formulation has a pH inthe range from about 7.0 to about 8.0.

In yet a further embodiment of the invention, the formulation has a pHin the range from about 7.2 to about 8.0.

In a further embodiment of the invention, the formulation has a pH inthe range from about 7.0 to about 8.3.

In yet a further embodiment of the invention, the formulation has a pHin the range from about 7.3 to about 8.3.

In a preferred embodiment of the invention, the formulations contain, inaddition to a peptide and propylene glycol, a buffer and/or apreservative.

Where a buffer is to be included in the formulations of the invention,the buffer is selected from the group consisting of sodium acetate,sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine,arginin, sodium dihydrogen phosphate, disodium hydrogen phosphate,sodium phosphate, and tris(hydroxymethyl)-aminomethan, or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention. In a preferred embodiment of the inventionthe buffer is glycylglycine, sodium dihydrogen phosphate, disodiumhydrogen phosphate, sodium phosphate or mixtures thereof.

Where a pharmaceutically acceptable preservative is to be included inthe formulations of the invention, the preservative is selected from thegroup consisting of phenol, m-cresol, methyl p-hydroxybenzoate, propylp-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, ormixtures thereof. Each one of these specific preservatives constitutesan alternative embodiment of the invention. In a preferred embodiment ofthe invention the preservative is phenol or m-cresol.

In a further embodiment of the invention the preservative is present ina concentration from about 0.1 mg/ml to about 50 mg/ml, more preferablyin a concentration from about 0.1 mg/ml to about 25 mg/ml, and mostpreferably in a concentration from about 0.1 mg/ml to about 10 mg/ml

The use of a preservative in pharmaceutical compositions is well-knownto the skilled person. For convenience reference is made to Remington:The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention the formulation may furthercomprise a chelating agent where the chelating agent may be selectedfrom salts of ethlenediaminetetraacetic acid (EDTA), citric acid, andaspartic acid, and mixtures thereof. Each one of these specificchelating agents constitutes an alternative embodiment of the invention.

In a further embodiment of the invention the chelating agent is presentin a concentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment ofthe invention the chelating agent is present in a concentration from 0.1mg/ml to 2 mg/ml. In a further embodiment of the invention the chelatingagent is present in a concentration from 2 mg/ml to 5 mg/ml.

The use of a chelating agent in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention the formulation may furthercomprise a stabiliser selected from the group of high molecular weightpolymers or low molecular compounds where such stabilizers include, butare not limited to, polyethylene glycol (e.g. PEG 3350),polyvinylalcohol (PVA), polyvinylpyrrolidone, carboxymethylcellulose,different salts (e.g. sodium chloride), L-glycine, L-histidine,imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan,threonine and mixtures thereof. Each one of these specific stabilizersconstitutes an alternative embodiment of the invention. In a preferredembodiment of the invention the stabiliser is selected from the groupconsisting of L-histidine, imidazole and arginine.

In a further embodiment of the invention the high molecular weightpolymer is present in a concentration from 0.1 mg/ml to 50 mg/ml. In afurther embodiment of the invention the high molecular weight polymer ispresent in a concentration from 0.1 mg/ml to 5 mg/ml. In a furtherembodiment of the invention the high molecular weight polymer is presentin a concentration from 5 mg/ml to 10 mg/ml. In a further embodiment ofthe invention the high molecular weight polymer is present in aconcentration from 0 mg/ml to 20 mg/ml. In a further embodiment of theinvention the high molecular weight polymer is present in aconcentration from 20 mg/ml to 30 mg/ml. In a further embodiment of theinvention the high molecular weight polymer is present in aconcentration from 30 mg/ml to 50 mg/ml.

In a further embodiment of the invention the low molecular weightcompound is present in a concentration from 0.1 mg/ml to 50 mg/ml. In afurther embodiment of the invention the low molecular weight compound ispresent in a concentration from 0.1 mg/ml to 5 mg/ml.

In a further embodiment of the invention the low molecular weightcompound is present in a concentration from 5 mg/ml to 10 mg/ml. In afurther embodiment of the invention the low molecular weight compound ispresent in a concentration from 10 mg/ml to 20 mg/ml. In a furtherembodiment of the invention the low molecular weight compound is presentin a concentration from 20 mg/ml to 30 mg/ml. In a further embodiment ofthe invention the low molecular weight compound is present in aconcentration from 30 mg/ml to 50 mg/ml.

The use of a stabilizer in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention the formulation of theinvention may further comprise a surfactant where a surfactant may beselected from a detergent, ethoxylated castor oil, polyglycolyzedglycerides, acetylated monoglycerides, sorbitan fatty acid esters,poloxamers, such as 188 and 407, polyoxyethylene sorbitan fatty acidesters, polyoxyethylene derivatives such as alkylated and alkoxylatedderivatives (tweens, e.g. Tween-20, or Tween-80), monoglycerides orethoxylated derivatives thereof, diglycerides or polyoxyethylenederivatives thereof, glycerol, cholic acid or derivatives thereof,lecithins, alcohols and phospholipids, glycerophospholipids (lecithins,kephalins, phosphatidyl serine), glyceroglycolipids (galactopyransoide),sphingophospholipids (sphingomyelin), and sphingoglycolipids (ceramides,gangliosides), DSS (docusate sodium, docusate calcium, docusatepotassium, SDS (sodium dodecyl sulfate or sodium lauryl sulfate),dipalmitoyl phosphatidic acid, sodium caprylate, bile acids and saltsthereof and glycine or taurine conjugates, ursodeoxycholic acid, sodiumcholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate,N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic(alkyl-aryl-sulphonates)monovalent surfactants, palmitoyllysophosphatidyl-L-serine, lysophospholipids (e.g.1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine), alkyl, alkoxyl(alkyl ester), alkoxy(alkyl ether)-derivativesof lysophosphatidyl and phosphatidylcholines, e.g. lauroyl and myristoylderivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine,and modifications of the polar head group, that is cholines,ethanolamines, phosphatidic acid, serines, threonines, glycerol,inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP,lysophosphatidylserine and lysophosphatidylthreonine, zwitterionicsurfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate,dodecylphosphocholine, myristoyl lysophosphatidylcholine, hen egglysolecithin), cationic surfactants (quarternary ammonium bases) (e.g.cetyltrimethylammonium bromide, cetylpyridinium chloride), non-ionicsurfactants, polyethyleneoxide/polypropyleneoxide block copolymers(Pluronics/Tetronics, Triton X-100, Dodecyl β-D-glucopyranoside) orpolymeric surfactants (Tween-40, Tween-80, Brij-35), fusidic acidderivatives—(e.g. sodium tauro-dihydrofusidate etc.), long-chain fattyacids and salts thereof C6-C12 (eg. oleic acid and caprylic acid),acylcarnitines and derivatives, N^(α)-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N^(α)-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, N^(α)-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, or thesurfactant may be selected from the group of imidazoline derivatives, ormixtures thereof. Each one of these specific surfactants constitutes analternative embodiment of the invention. The use of a surfactant inpharmaceutical compositions is well-known to the skilled person. Forconvenience reference is made to Remington: The Science and Practice ofPharmacy, 19^(th) edition, 1995.

The formulations of the invention may be prepared by conventionaltechniques, e.g. as described in Remington's Pharmaceutical Sciences,1985 or in Remington: The Science and Practice of Pharmacy, 19^(th)edition, 1995, where such conventional techniques of the pharmaceuticalindustry involve dissolving and mixing the ingredients as appropriate togive the desired end product.

As mentioned above, in a preferred embodiment, the formulations of theinvention-contain, in addition to a peptide and propylene glycol, abuffer and/or a preservative.

In one embodiment, the method for preparing such a peptide formulationcomprises:

-   -   a) preparing a first solution by dissolving preservative,        propylene glycol and buffer in water;    -   b) preparing a second solution by dissolving the peptide in        water;    -   c) mixing the first and second solutions; and    -   d) adjusting the pH of the mixture in c) to the desired pH.

In another embodiment, the method for preparing such a peptideformulation comprises:

-   -   a) preparing a first solution by dissolving preservative and        buffer in water;    -   b) adding propylene glycol to the first solution;    -   c) mixing the first solution with a second solution containing        peptide dissolved in water; and    -   d) adjusting the pH of the mixture in c) to the desired pH.

In yet another embodiment, the method for preparing a peptideformulation comprises:

-   -   a) preparing a solution by dissolving preservative, buffer and        propylene glycol in water;    -   b) adding the peptide to the solution of step a); and    -   c) adjusting the pH of the solution of step b) to the desired        pH.

As the formulations of the invention are optimal for production and foruse in injection devices since they exhibit reduced deposits ofproduction equipment and reduced clogging of injection devices, theabove methods of production can be used to produce peptide formulationssuitable for use in production and/or for use in injection devices.

The formulations of the invention are suitable for administration to amammal, preferably a human. The route of administration of theformulations of the invention may be any route which effectivelytransports the peptide contained in the formulation to the appropriateor desired site of action, such as oral, nasal, buccal, pulmonal,transdermal or parenteral.

Due to the ability of propylene glycol to reduce clogging of injectiondevices when compared to other isotonic agents and to mannitol inparticular, in a preferred embodiment, the formulations of the inventionare to be administered parenterally to a patient in need thereof.Parenteral administration may be performed by subcutaneous,intramuscular or intravenous injection by means of a syringe, optionallya pen-like syringe. Alternatively, parenteral administration can beperformed by means of an infusion pump.

A further option is a composition which may be a powder or a liquid forthe administration of the formulation in the form of a nasal or pulmonalspray. As a still further option, the formulation can also beadministered transdermally, e.g. from a patch, optionally aiontophoretic patch, or transmucosally, e.g. bucally. Theabove-mentioned possible ways to administer the formulations of theinvention are not to be considered as limiting the scope of theinvention.

Of course, it is understood that depending on the peptide or peptidesincluded in the formulations of the invention, the formulations may beused in methods of treatment of diseases or conditions for which use ofthe peptide is indicated. One skilled in the art would understand thatwhen used in such methods of treatment, the formulations would have tobe administered in amount effective to treat the condition or diseasefor which the peptide was being administered where an “effective amount”or an “amount . . . effective” is understood to mean a dosage which issufficient in order for the treatment of the patient with the disease orcondition to be treated to be effective compared to treatment withoutthe administered dosage. It is to be understood that “an effectiveamount” is the effective dose to be determined by a qualifiedpractitioner, who may titrate dosages to achieve the desired response.Factors for consideration of dose will include potency, bioavailability,desired pharmacokinetic/pharmacodynamic profiles, the condition ordisease to be treated (e.g. diabetes, obesity, weight loss, gastriculcers), patient-related factors (e.g. weight, health, age, etc.),presence of co-administered medications (e.g. insulin), time ofadministration, or other factors known to a medical practitioner.

The present invention also relates to a method for reducing deposits onproduction equipment during production of a peptide formulation, wherethe method comprises replacing the isotonicity agent previously utilizedin said formulation with propylene glycol at a concentration of between1-100 mg/ml.

In one embodiment, the reduction in deposits on the production equipmentduring production by the propylene glycol-containing formulationrelative to that observed for the formulation containing the previouslyutilized isotonicity agent is measured by a simulated filling experimentas described in the Examples.

In another embodiment, the isotonicity agent to be replaced by propyleneglycol is selected from the group consisting of sorbitol, sucrose,glycine, mannitol, lactose monohydrate, arginin, myo-inositol anddimethylsulfon.

In a further embodiment, the isotonicity agent previously utilized insaid formulation is replaced with propylene glycol in a concentration offrom about 1 to about 50 mg/ml.

In another embodiment, the isotonicity agent previously utilized in saidformulation is replaced with propylene glycol in a concentration of fromabout 5 to about 25 mg/ml.

In yet another embodiment, the isotonicity agent previously utilized insaid formulation is replaced with propylene glycol in a concentration offrom about 8 to about 16 mg/ml.

In another embodiment of the invention, the propylene glycol-containingformulation has a pH in the range from about 7.0 to about 9.5.

In a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from about 7.0 toabout 8.0.

In yet a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from 7.2 to about8.0.

In a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from about 7.0 toabout 8.3.

In a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from 7.3 to about8.3.

The present invention also relates to a method for reducing deposits inthe final product during production of a peptide formulation, where themethod comprises replacing the isotonicity agent previously utilized insaid formulation with propylene glycol at a concentration of between1-100 mg/ml.

In one embodiment, the reduction in deposits in the final product ismeasured by a reduction in the number of vials and/or cartridges of thepropylene glycol-containing formulation that must be discarded due todeposits relative to number of vials and/or cartridges of theformulation containing the previously utilized isotonicity agent thatmust be discarded due to deposits.

In another embodiment, the isotonicity agent to be replaced by propyleneglycol is selected from the group consisting of sorbitol, sucrose,glycine, mannitol, lactose monohydrate, arginin, myo-inositol anddimethylsulfon.

In a further embodiment, the isotonicity agent previously utilized insaid formulation is replaced with propylene glycol in a concentration offrom about 1 to about 50 mg/ml.

In another embodiment, the isotonicity agent previously utilized in saidformulation is replaced with propylene glycol in a concentration of fromabout 5 to about 25 mg/ml.

In yet another embodiment, the isotonicity agent previously utilized insaid formulation is replaced with propylene glycol in a concentration offrom about 8 to about 16 mg/ml.

In another embodiment of the invention, the propylene glycol-containingformulation has a pH in the range from about 7.0 to about 9.5.

In a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from about 7.0 toabout 8.0.

In yet a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from 7.2 to about8.0.

In a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from about 7.0 toabout 8.3.

In a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from 7.3 to about8.3.

The present invention further relates to a method for reducing theclogging of injection devices by a peptide formulation, where the methodcomprises replacing the isotonicity agent previously utilized in saidformulation with propylene glycol at a concentration of between 1-100mg/ml.

In one embodiment, the reduction in clogging of the injection device bythe propylene glycol-containing formulation relative to that observedfor the formulation containing the previously utilized isotonicity agentis measured in a simulated in use study as described in the Examples.

In another embodiment, the isotonicity agent to be replaced by propyleneglycol is selected from the group consisting of inositol, maltose,glycine, lactose and mannitol.

In a further embodiment, the isotonicity agent previously utilized insaid formulation is replaced with propylene glycol in a concentration offrom about 1 to about 50 mg/ml.

In another embodiment, the isotonicity agent previously utilized in saidformulation is replaced with propylene glycol in a concentration of fromabout 5 to about 25 mg/ml.

In yet another embodiment, the isotonicity agent previously utilized insaid formulation is replaced with propylene glycol in a concentration offrom about 8 to about 16 mg/ml.

In another embodiment of the invention, the propylene glycol-containingformulation has a pH in the range from about 7.0 to about 9.5.

In a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from about 7.0 toabout 8.0.

In yet a further embodiment of the invention, the propyleneglycol-containing formulation has a pH in the range from 7.2 to about8.0.

All scientific publications and patents cited herein are specificallyincorporated by reference. The following examples illustrate variousaspects of the invention but are in no way intended to limit the scopethereof.

EXAMPLES Example 1 Simulated Filling Experiments, Drop and CloggingTests of Replacement Candidates for Mannitol

As laboratory experiments have shown that with regards to clogging ofneedles and deposits on needles, formulations without peptide(“placebo”) give the same conclusions as formulations with peptide at0.3-5.0 mg/ml, the screening studies in Example 1 have been done usingplacebo except where indicated otherwise.

Preparation of Formulations with Different Isotonic Agents

Preservative (5.5 mg/ml phenol) and buffer 1.24 mg/ml disodium hydrogenphosphate, dihydrate) were dissolved in water and the isotonic agent wasadded while stirring. pH was adjusted to pH 7.9 using Sodium Hydroxideand/or Hydrochloric acid. Finally, the formulation was filtered througha 0.22 μm filter. The isotonic agents tested in each formulation andtheir concentrations are shown in Table 1.

TABLE 1 Composition of the tested formulations Formulation no. Tonicitymodifier 1 Glucose monohydrate (38.0 mg/ml) 2 Laktose monohydrate (65.0mg/ml) 3 Maltose (67.2 mg/ml) 4 Glycine (15.1 mg/ml) 5 Polyethylenglycol400 (77.5 mg/ml) 6 L-arginin (24.6 mg/ml) 7 Myo-Inositol (35.2 mg/ml) 8Propylene glycol (13.7 mg/ml) 9 Dimethylsulfon (18 mg/ml) 10 Mannitol(35.9 mg/ml) 11 Sorbitol (39.5 mg/ml) 12 Xylitol (39.5 mg/ml) 13 Sucrose(79.1 mg/ml 14 Glycerol (16 mg/ml)

Osmolarity

The osmolarity of the different placebo formulations was determined andthe results are shown in Table 2.

An isotonic solution has an osmolarity of around 0.286 osmol/L. As canbe seen from Table 2 three of the formulations (PEG 400, sucrose andxylitol) are more than 20% from being isotonic (0.229-0.343 osmol/l),however for these kind of experiments the osmolarity is not expected toinfluence the results, though, the tonicity of the formulations shouldbe adjusted in future experiments.

TABLE 2 The measured osmolarity of the formulations Formulation no.Isotonic agent Osmolarity 1 Glucose monohydrate (38.0 mg/ml) 0.315 2Laktose monohydrate (65.0 mg/ml) 0.283 3 Maltose (67.2 mg/ml) 0.306 4Glycine (15.1 mg/ml) 0.286 5 Polyethylenglykol 400 (77.5 mg/ml) 0.370 6L-arginin(24.6 mg/ml) 0.318 7 Myo-Inositol (35.2 mg/ml) 0.285 8Propylene glycol (13.7 mg/ml) 0.268 9 Dimethylsulfon (18 mg/ml) 0.274 10Mannitol (35.9 mg/ml) 0.284 11 Sorbitol (39.5 mg/ml) 0.310 12 Xylitol(39.5 mg/ml) 0.351 13 Sucrose (79.1 mg/ml 0.346 14 Glycerol (16 mg/ml)0.262

Drop Test

A droplet of each formulation is placed on a microscope slide and let todry. The deposit is visually examined by eye and light microscope.

A photograph of the dried droplets of some of the formulations is shownin FIG. 1. In this figure it is clearly observed that mannitol causedeposits on the microscope slide when let to dry. No deposits wereobserved for sorbitol, xylitol, sucrose and glycerol. The droplet on thefar right (Form 1) contains mannitol andArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37).

In FIG. 2, the candidates causing the most deposits on the microscopeslide are shown. For comparison glycerol, which does not cause deposits,is shown (mannitol, arginine, inositol).

Clogging Test

In this test 10 NovoPens® 1.5 ml mounted with NovoFine 30® G (G 30needle) were tested for each formulation, 5 of them placed in uprightand 5 in horizontal position. The Pensystems were stored at roomtemperature in between testing. Each day the needle was examined fordeposits and an air shot was performed prior to injection into a tissue.Degree of resistance and clogging, if any, was noted. Injections weremade on a daily basis with the same needle, and this was done for 9working days for all the formulations.

The results from the clogging test are shown in Table 3.

TABLE 3 Clogging test in NovoPen 1.5 using 30 G NovoFine Isotonic agentDrop at Dried Gel-like (no. of Some Much top of drop at drop on Depositsobservations) resistance Resistance resistance Clogged needle needle topneedle on needle Mannitol (90) 10 0 0 0 0 2 0 43  Glycerol (90) 13 0 0 01 0 3 0 Sucrose (90) 23 0 0 0 0 0 21 0 Propylene 20 0 0 0 0 0 0 0 glycol(90) PEG 400 (90) 25 1 0 0 12 (5 at 0 0 0 needle) arginin (90) 26 2 0 03 (2 at 1 0 0 needle) Xylitol (90) 14 0 0 0 5 0 0 0 Dimethylsulfon 21 00 0 4 0 0 0 (90) sorbitol (90) 12 0 0 0 9 1 0 1 Myo-inositol 20 1 2 6 60 0 47  (90) Glucose (90) 32 11 5 0 16 (7 at 1 0 (1 at needle) needle)glycine (90) 41 9 2 0 1 (2 at 0 0 31 (2 at needle) needle) maltose (90)35 8 7 4 16 (6 at 0 0 1 (5 at needle) needle) laktose (90) 44 10 8 0 5 00 31 (2 at needle)

In Table 3 and in FIG. 3 it was observed that inositol and maltoseclogged the needle. For comparison glycerol which does not clog theneedle is shown in FIG. 3. In FIG. 4, and in Table 3, it was observedthat formulations containing glycine, lactose and mannitol gave rise toa lot of deposits on the needle. For glycine, the deposits were adroplet deposited down the needle, whereas for lactose and mannitol thedeposits occurred at the top of the needle.

Simulated Filling

1 L of each formulation was subjected to a simulated filling experimentwhich lasted for 24 hours. After 24 hours the filling equipment wasinspected for the presence of deposits. Based on the results from thesimulated filling studies (data not shown), the placebo formulations canbe divided into three categories. 1. Those isotonic agents that do notcause deposits on the filling equipment: Xylitol, glycerol, glucosemonohydrate, maltose, PEG 400 and propylene glycol. 2. Those isotonicagent that cause few deposits and have superior filling propertiescompared to mannitol: Sorbitol, sucrose and glycine. 3. Those isotonicagent that are comparable or worse than mannitol: Mannitol, lactosemonohydrate, arginin, myo-inositol and dimethylsulfon.

Conclusion

In the simulated filling experiment xylitol, glycerol, glucose, maltose,PEG 400, propylene glycol, sorbitol, sucrose and glycine were found tobe suitable replacements candidates for mannitol. However, as glucose isa reducing saccharide, and therefore is able to initiate unwanteddegradation in the formulation, this tonicity modifier is ruled out.Furthermore, maltose is ruled out due to clogging of needles. This leadsto the following candidates: glycerol, xylitol, sorbitol, sucrose,glycine, propylene glycol and PEG 400, which are found to have suitableproperties as replacements candidates for mannitol in peptideformulations with regards to drop test, clogging of needles andsimulated filling.

However, on the basis of the following considerations, propylene glycolwas chosen as the isotonic agent over the other candidates to be furtherinvestigated in head to head comparison studies with mannitol:

-   -   a. propylene glycol was observed to have no influence on the        physical and chemical stability of        Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37)-containing        formulations;    -   b. propylene glycol was observed to have no influence on        antimicrobial preservative testing; and    -   c. use of propylene glycol would no require that further        toxicity studies be tested

Example 2 Comparison of Mannitol and Propylene Glycol-Containing PlaceboFormulations in Simulated Filling Studies and Simulated Use StudiesPreparation of Formulations

Preservative and buffer were dissolved in water and the isotonic agentwas added while stirring. pH was adjusted to the aimed pH using SodiumHydroxide and/or Hydrochloric acid. Finally, the formulation wasfiltered through a 0.22 μm filter. The compositions of the formulationswere as follows:

-   -   Disodium hydrogen phosphate, dihydrate: 1.42 mg/ml    -   Phenol: 5.5 mg/ml    -   Propylene glycol or mannitol: 13.7 or 35.9 mg/ml    -   Water for Injection: up to 1.0 ml.    -   pH: 7.90

Simulated Filling Study

A simulated filling study lasting 24 hours was performed as described inExample 1 and after 24 hours, the filling equipment was inspected forthe presence of deposits. No deposits were observed on the fillingequipment for the propylene glycol formulation. By comparison, after 24hours, a lot of deposits were observed on the filling equipment for themannitol formulation (see FIG. 6).

Simulated in Use Study

For the simulated in use study, a clogging test was conducted asdescribed in Example 1. The same needle was used during the study periodof ten working days and each day, the needle was inspected for thepresence of deposits. FIG. 7 shows photographs of needles dosed with thepropylene glycol (top panel) or mannitol (bottom panel) containingformulations. Deposits on the needle were observed in 48% of the caseswhen mannitol was used as an isotonic agent whereas no deposits wereobserved when propylene glycol was used as the isotonic agent.

Example 3 Comparison of Propylene Glycol to Mannitol InArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) ContainingFormulations Preparation of Formulations

Preservative, isotonic agent (mannitol or propylene glycol) and bufferwere dissolved in water and pH was adjusted to the desired pH.Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) was dissolvedin water while stirring slowly. The two solutions were then mixed and pHadjusted to the desired pH using sodium hydroxide and/or hydrochloricacid. Finally, the formulation was filtered through a 0.22 μm filter.The compositions of the formulations were as follows:

Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) (6.25 mg/ml),

Disodium hydrogen phosphate, dihydrate (1.42 mg/ml),

Phenol (5.5 mg/ml),

mannitol or propylene glycol (35.9 or 14.0 mg/ml),

Water for Injection (up to 1.0 ml),

pH: 8.15

Simulated in Use Study

For the simulated in use study, a clogging test was conducted asdescribed in Example 1 except that a G31 needle was used. The same G31needle was used during the study period of ten working days and eachday, the needle was inspected for the presence of deposits. FIG. 7 showsphotographs of needles with no deposits when dosed with the propyleneglycol (bottom panel) or showing deposits when dosed with the mannitol(top panel) containing formulations.

For the mannitol containing formulation, clogging of the needle wasobserved in 1 out of 10 cases on day 4, 2 out of 10 cases on day 5, 3out of 10 cases on day 8 and 4 out of 10 cases on day 9. By comparison,no clogging of needles was observed for the propylene glycol containingformulation.

It is believed that similar results to those obtained with theabove-described propylene glycol-containing formulation would also beobtained if the pH was adjusted to 7.40, 7.70 or 7.90. In addition,additional formulations which could be tested include those having thefollowing compositions:

Buffering agents: glycylglycine (1.32 mg/ml), L-Histidine (1.55 mg/ml),Hepes (2.38 mg/ml), or bicine (1.63 mg/ml)

Preservatives: phenol (5.0 or 5.5 mg/ml), benzylalcohol (18 mg/ml) or amixture of m-cresol and phenol (2.5/2.0 mg/ml)

Propylene glycol: 14.0 or 14.3 mg·ml

Water for injection: up to 1.0 ml

pH: 7.40, 7.70, 7.90 or 8.15

Example 4 Influence of Peptide Concentration on Clogging of Needles

Arg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37) formulationswere prepared as described in Example 3 using peptide concentrationsranging from 0-5 mg/ml ofArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37). Thecompositions of the formulations were as follows:

Liraglutide: 0, 0.3, 3 and 5 mg/mlDisodium hydrogen phosphate, dihydrate: 0.71 mg/mlSodium dihydrogenphosphate, dihydrate: 0.62 mg/mlMannitol: 36.9 mg/mlPhenol: 5.0 mg/mlWater for injection: up to 1.0 mlpH 7.40

A simulated in use study was conducted as in Example 3 except that a G30needle was used and the results (data not shown) indicated that theclogging effect of the mannitol-containing formulations relative to theabsence of clogging with the propylene glycol formulations was observedindependent of the peptide concentration.

Example 5 Clogging of needles in Lys β29 (Nε-tetradecanoyl) des(B30)human insulin and NovoMix 30 formulations Containing MannitolPreparation of Formulations

The Lys β29 (Nε-tetradecanoyl) des(B30) human insulin-containingformulation was prepared as follows:

a) Prepared a first solution by dissolving buffer, sodium chloride,preservatives (phenol and m-cresol) and mannitol in waterb) Prepared a second solution of Lys β29 (Nε-tetradecanoyl) des(B30)human insulin and zinc acetate dissolved in waterc) added the peptide-containing solution of step b) to the solution ofstep a); andd) adjusted the pH of the solution to the desired pH

The composition of Lys β29 (Nε-tetradecanoyl) des(B30) humaninsulin-containing formulation prepared in the above manner was asfollows:

Lys β29 (Nε-tetradecanoyl) des(B30) human insulin (2400 nmol), Phenol(1.80 mg/ml), m-cresol (2.06 mg/ml), Mannitol (30.0 mg/ml),disodiumphosphate, dihydrate (0.890 mg/ml), Sodium chloride (1.17mg/ml), Zinc acetate (65.4 ug/ml), water for injection (up to 1.0 ml),pH: 7.4

The NovoMix 30-containing formulation was prepared as follows:

a) Prepared a solution by dissolving buffer, sodium chloride, phenol,mannitol and sodium hydroxide in waterb) Prepared a solution of sodium chloride, phenol and mannitol in waterc) Prepared a solution of protamine sulphate in waterd) Prepared a solution of insulin, hydrochloric acid and zinc in watere) Solutions b), c) and d) were mixedf) Solution e) was added to the solution of step a)g) Adjusted the pH of the solution to the desired pH and crystallized atroom temperatureh) Prepared a solution by dissolving m-cresol, phenol and mannitol inwateri) Solution h) is added to the crystalline fraction of step g); andj) Adjusted the pH to the desired pH

The composition of the NovoMix 30-containing formulation prepared in theabove manner was as follows:

Insulin aspart (100 units/ml), protamine sulphate (approx. 0.33 mg/ml),phenol (1.50 mg/ml), m-cresol (1.72 mg/ml), mannitol (30.0 mg/ml),disodiumphosphate dihydrate (1.25 mg/ml), sodium chloride (0.58 mg/ml),zinc (19.6 ug/ml), water for injection (up to 1.0 ml), pH: 7.3.

Results

A simulated in use study was conducted as described in Example 3 usingG31 needles where 20 needles were investigated for 10 days. The resultswere as follows: Clogging of needles was observed for Lys β29(Nε-tetradecanoyl) des(B30) human insulin on day 2 (5%), day 3 (70%) andon day 4 (100%). Clogging of needles for NovoMix 30 was observed on day3 (5%), day 4 (10%), day 5 (35%), day 6 (40%), day 8 (50%), day 9 (55%)and day 10 (80%). Thus, the effect of mannitol on the clogging ofneedles is independent of the type of peptide included in theformulations since a comparable clogging effect was observed withArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37), Lys β29(Nε-tetradecanoyl) des(B30) human insulin and NovoMix 30.

Example 6 Testing of Lys β29 (Nε-tetradecanoyl) des(B30) human insulinand NovoMix 30 formulations containing propylene glycol

The preparation and composition of the Lys β29 (Nε-tetradecanoyl)des(B30) human insulin and NovoMix 30 formulations will be as describedin Example 5 except that mannitol will be replaced with a concentrationof propylene glycol that assures tonicity. A simulated in use test willthen be conducted as described in Example 5.

Based on the fact that the clogging effect of Lys β29 (Nε-tetradecanoyl)des(B30) human insulin and NovoMix 30 mannitol-containing formulationswas similar to that observed withArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37)mannitol-containing formulations, it is believed that the effect ofpropylene glycol on the clogging effect of Lys β29 (Nε-tetradecanoyl)des(B30) human insulin and NovoMix 30-containing formulations will besimilar to that observed withArg³⁴,Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37)-containingformulations.

1. A pharmaceutical formulation comprising at least one peptide andpropylene glycol, wherein said propylene glycol is present in saidformulation in a final concentration of from about 1 mg/ml to about 100mg/ml and wherein said formulation has a pH of from about 7.0 to about10.0.
 2. The formulation according to claim 1, wherein the concentrationof propylene glycol is from about 1 mg/ml to about 50 mg/ml.
 3. Theformulation according to claim 1, wherein the concentration of propyleneglycol is from about 5 mg/ml to about 25 mg/ml.
 4. The formulationaccording to claim 1, wherein the concentration of propylene glycol isfrom about 8 mg/ml to about 16 mg/ml.
 5. The formulation according toclaim 1, wherein the pH of said formulation is about 7.0 to about 9.5.6. The formulation according to claim 1, wherein the pH of saidformulation is about 7.0 to about 8.3.
 7. The formulation according toclaim 1, wherein the pH of said formulation is about 7.3 to about 8.3.8. The formulation according to claim 1, further comprising apreservative.
 9. The formulation according to claim 8, wherein saidpreservative is present in a concentration from 0.1 mg/ml to 20 mg/ml.10. The formulation according to claim 1, further comprising a buffer.11. The formulation according to claim 10, wherein said buffer isselected from the group consisting of glycylglycine, L-histidine, Hepes,bicine and disodium phosphate dihydrate.
 12. The formulation accordingto claim 1, wherein said peptide is a GLP-1 agonist.
 13. The formulationaccording to claim 12, wherein said GLP-1 agonist is selected from thegroup consisting of GLP-1(7-36)-amide, GLP-1(7-37), a GLP-1(7-36)-amideanalogue, a GLP-1(7-37) analogue, or a derivative of any of these. 14.The formulation according to claim 13, wherein said GLP-1 agonist is aderivative of GLP-1(7-36) or GLP-1(7-37) or a GLP-1(7-36)-amide analogueor a GLP-1(7-37) analogue, where said derivative has a lysine residueand a lipophilic substituent attached with or without a spacer to theepsilon amino group of said lysine.
 15. The formulation according toclaim 14, wherein said lipophilic substituent has from 8 to 40 carbonatoms.
 16. The formulation according to claim 15, wherein said spacer isan amino acid.
 17. The formulation according to claim 16, wherein saidGLP-1 agonist is Arg³⁴,Lys²⁶(N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37).18. The formulation according to claim 12, wherein said GLP-1 agonist isselected from the group consisting of Gly⁸-GLP-1(7-36)-amide,Gly⁸-GLP-1(7-37), Val⁸-GLP-1(7-36)-amide, Val⁸-GLP-1(7-37),Val⁸Asp²²-GLP-1(7-36)-amide, Val⁸Asp²²-GLP-1(7-37),Val⁸Glu²²-GLP-1(7-36)-amide, Val⁸Glu²²-GLP-1(7-37),Val⁸Lys²²-GLP-1(7-36)-amide, Val⁸Lys²²-GLP-1(7-37),Val⁸Arg²²-GLP-1(7-36)-amide, Val⁸Arg²²-GLP-1(7-37),Val⁸His²²-GLP-1(7-36)-amide, Val⁸His²²-GLP-1(7-37), Arg34GLP-1(7-37);Arg26,34,Lys36GLP-1(7-36); Arg26GLP-1(7-37); andGly8,Arg26,34,Glu37,Lys38GLP-1(7-38) analogues thereof and derivativesof any of these.
 19. The formulation according to claim 1, wherein saidpeptide is selected from insulin, an insulin analogue, a derivative ofinsulin or an insulin analogue or a mixture of any of the foregoing. 20.The formulation according to claim 19, wherein said peptide is aninsulin derivative in which said derivative contains a lysine residue towhich a lipophilic substituent of at least six carbon atoms is attached.21. The formulation according to claim 20, wherein the insulinderivative is Lys β29 (Nε-tetradecanoyl) des(B30) human insulin.
 22. Theformulation according to claim 20, wherein said insulin derivative isN^(□B29)-octanoyl insulin.
 23. The formulation according to claim 19,wherein said peptide is a 30/70 mixture of insulin aspart and insulinaspart protamine.
 24. The formulation according to claim 1, wherein saidpeptide is human growth hormone (hGH) or Met-hGH.
 25. The formulationaccording to claim 24, wherein said peptide is exendin 4, an exendin 4analogue or a derivative of exendin 4 or an exendin 4 analogue.
 26. Theformulation according to claim 25, wherein said peptide is exendin 4.27. The formulation according to claim 25, wherein said peptide isHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-amide.